Conformation of coenzyme pyrroloquinoline quinone and role of Ca ²⁺ in the catalytic mechanism of quinoprotein methanol dehydrogenase

Abstract

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca 2؉ , (PQQH)Ca 2؉ , and (PQQH2)Ca 2؉ complexes as well as the x-ray structure of (PQQ)Ca 2؉ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca 2؉ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca 2؉ , nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization We have had an extended interest in the chemistry of 2,7,9-tricarboxypyrroloquinoline quinone (pyrroloquinoline quinone, PQQ, and the older name methoxatin) and the mechanism of amine oxidation by PQQ (Structure 1) (1-5).Methanol dehydrogenase (MDH; EC 1.1.99.8) is a quinoprotein that requires Ca 2ϩ as well as PQQ cofactor (6-8) for activity in the oxidation of methanol to formaldehyde (9-15). Three-dimensional structures of MDH from methylotropic bacteria have been solved independently by two groups (16)(17)(18)(19). These crystal structures provide detailed information regarding the interaction between . According to these crystal structures, Ca 2ϩ binds to PQQ through N-6, C-5 carbonyl oxygen, and C-7 carboxylate. Protein Glu and Asn side chains occupy the remaining Ca 2ϩ coordination sites. The protein structure must be responsible for ligation of Ca 2ϩ to the N-6, C-5 carbonyl oxygen, and C-7 carboxylate functions of PQQ. Thus, in water the ligating agent ␣-picolinate (pyridine-2-carboxylate) has little affinity for Ca 2ϩ , whereas transition metals are ligated by the pyridine nitrogen and carboxylate oxygen. A number of possible mechanisms have been proposed for MDH over the years (13)(14)(15). Several of these proposed mechanisms have been proved to be invalid when the x-ray crystal structures became available. Two frequently mentioned mechanisms are shown in Scheme 1. The first mechanism involves a general base (Asp-303-CO 2 Ϫ ) catalyzed addition of methanol to the PQQ carbonyl group at C5 followed by a retro-ene reaction and the second mechanism involves general base (Asp-303-CO 2 Ϫ ) catalyzed proton abstraction concerted with hydride transfer. The retro-ene reaction in the first mechanism is not expected to be facile. Thus, retro-ene reactions are normally extremely slow and are generally carried out at high temperature (21,22).In this study we provide a theoretical approach to evaluate the role of Ca 2ϩ in the catalytic mechanism of MDH using an active site model based on the coordinates of the PQQ͞Ca 2ϩ ͞ MDH structure. In the course of the study we investigated the structure of PQQ in its different oxidation states and how its structure and conformation change ...

Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

“…Thus, Ca 2ϩ plays a catalytic role as in the oxidation of CH 3 OH 3 CH 2 O. This finding is in accord with Ca 2ϩ polarizing O5 of PQQ (11). It is to be expected that the interaction of Ba 2ϩ with the developing negative charge at the TS would be greater than that of Ca 2ϩ .…”

“…1-4). More recently, our attention has been directed to the employment of computational methods, particularly in the mechanism of the oxidation of methanol and glucose by the appropriate quinoprotein enzymes (5)(6)(7)(8)(9)(10)(11).…”

Structure of the Pyrroloquinoline Quinone Radical in Quinoprotein Ethanol Dehydrogenase

“…All three structures converged, and apart from the carboxylic group at C-9, which was slightly twisted to form a better hydrogen bond with the proton at N-1, the geometries were approximately planar, in agreement with previous work (30,48). However, it is not our purpose here to discuss all the details of the structures; rather we have concentrated on comparing the experimental and theoretical hfcs.…”

“…As shown in Fig. 1, apart from one review (21), reduced PQQ is usually shown protonated at both O-4 and O-5, whereas the radical is depicted singly protonated at either O-4 (30,31) or O-5 (20,24,32), although a deprotonated radical was recently postulated (33). Knowledge of the protonation states is crucial if the ET and proton transfer pathways in both reoxidation steps are to be understood, because depending on the protonation states of the initial and final molecules, the reaction is either simple ET or must be accompanied by the release of a proton.…”

Direct hydride transfer in the reaction mechanism of quinoprotein alcohol dehydrogenases: a quantum mechanical investigation

“…18 Calcium is also involved in the oxidation of glucose by PQQ-containing glucose dehydrogenases. 19 In the soluble GDH isolated from Acinetobacter calcoaceticus, for which a high-resolution X-ray structure has recently become available, 20 a histidine is found to act as the activesite base.…”